Abstract
The mixed epidermal growth factor receptor (EGFR)-DNA targeting properties of SMA41, a 6-(3-methyl-1,2,3-triazen-1-yl)-4-anilinoquinazoline designed to release N4-m-tolyl-quinazoline-4,6-diamine henceforth referred to as SMA52 [an inhibitor of EGFR tyrosine kinase (TK)] and methyldiazonium (a DNA methylating species) were studied in the O6-methylguanine-DNA methyltransferase (MGMT)-proficient and high EGFR-expressing epidermoid carcinoma of the vulva cell line A431. The effects of SMA41 were compared with those of SMA52 alone, and temozolomide (TEM), a clinical prodrug of 5-(3-methyltriazen-1-yl)imidazole-4-carboxamide (MTIC) that is inactive in MGMT-proficient cells. The results showed that 1) the chimeric SMA41 could degrade in serum-containing medium (t1/2 of ∼30 min) to generate, as predicted, the free inhibitor SMA52 as the most abundant metabolite (∼81% yield); 2) in contrast to SMA52 alone, the chimeric SMA41 and TEM induced significant DNA damage in A431 cells after 30-min or 2-h drug exposures, as confirmed by alkaline single-cell gel microelectrophoresis (comet) assay; 3) SMA41 showed 5-fold greater affinity for the ATP binding site of EGFR than independently synthesized SMA52 in an enzyme assay and blocked EGF-induced tyrosine phosphorylation and EGFR autophosphorylation in A431 cells in a dose-dependent manner; 4) these mixed targeting properties of SMA41, combined with its ability to be converted to another potent EGFR TK inhibitor (e.g., SMA52) by hydrolytic cleavage, translated into over 8-fold greater antiproliferative activity than TEM, which showed no EGFR targeting properties (IC50 competitive binding >100 μM); 5) under continuous drug exposure (3–6-day sulforhodamine and clonogenic assays), SMA41 was almost equipotent with SMA52; however, in a short 2-h drug exposure followed by incubation in drug-free media, SMA52 showed an almost complete loss of antiproliferative activity over the whole dose range. In contrast, SMA41 retained almost 100% of its activity, indicating a more sustained growth inhibitory activity. The results in toto suggest that the superior antiproliferative activity of SMA41 may be due to a combination of events associated with its binary EGFR TK and DNA targeting properties.
Footnotes
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Send reprint requests to: Bertrand J. Jean-Claude, Ph.D., Cancer Drug Research Laboratory, Department of Medicine, Division of Medical Oncology, McGill University Health Center/Royal Victoria Hospital, 687 Pine Ave. West, Room M-719, Montreal, Quebec, Canada, H3A 1A1. E-mail: bertrand{at}med.mcgill.ca
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This study was supported by Cancer Research Society Inc. (CRS). This project was also partially supported by the Fonds pour la formation des Chercheurs et l'Aide a la Recherche (FCAR). S.L.M. is supported by a McGill University Faculty of Medicine Award.
- Abbreviations:
- EGFR
- epidermal growth factor receptor
- TK
- tyrosine kinase
- RTK
- receptor tyrosine kinase
- TEM
- temozolomide
- MTIC
- 5-(3-methyltriazen-1-yl)imidazole-4-carboxamide
- DMSO
- dimethyl sulfoxide
- SRB
- sulforhodamine B
- HPLC
- high pressure liquid chromatography
- PGT
- poly(l-glutamic acid-l-tyrosine, 4:1)
- PBS
- phosphate-buffered saline
- HRP
- horseradish peroxidase
- ELISA
- enzyme-linked immunosorbent assay
- EGF
- epidermal growth factor
- MGMT
- O6-methylguanine DNA methyl transferase
- Received September 25, 2000.
- Accepted November 29, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
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